Antitumor agents

ABSTRACT

The present invention provides compounds of general formula I or II:                    
     wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7  have any of the values defined in the specification, and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions comprising compounds of formula I or II, intermediates and processes useful for preparing compounds of formula I or II, and methods comprising inhibiting tumor growth or treating cancer by administering one or more compounds of formula I or II.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US99/03660, filed on Feb. 19, 1999; which is a continuation andclaims priority to U.S. Ser. No. 09/026,633, filed Feb. 20, 1998, nowissued as U.S. Pat. No. 6,025,328, all of which are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

A listing of human cancers for which chemotherapy has exerted aredominant role in increasing life span, approaching normal lifeexpectancy, includes Burkitt's lymphoma, acute lymphocytic leukemia andHodgkin's disease, along with about 10-15 other tumor types. Forexample, see A. Golden et al., Eur. J. Cancer, 17, 129 (1981) (Table 1).While the cure rate of these cancers illustrates the level of success ofscreening systems in selecting antitumor agents that are effective inman, these responsive tumors represent only a small fraction of thevarious types of cancer and, notably, there are relatively few drugshighly active against solid tumors such as ovarian cancer, breastcancer, lung cancer and the like. Such drugs include cyclophosphamide,adriamycin, 5-FU, hexamethylmelamine and the like. Thus, patients withmany types of malignancies remain at significant risk for relapse andmortality.

After relapse, some patients can be reinduced into remission with theirinitial treatment regimen. However, higher doses of the initialchemotherapeutic agent or the use of additional agents are frequentlyrequired, indicating the development of at least partial drugresistance. Recent evidence indicates drug resistance can developsimultaneously to several agents, including ones to which the patientwas not exposed. The development of multiple-drug resistant (mdr) tumorsmay be a function of tumor mass and constitutes a major cause oftreatment failure. To overcome this drug resistance, high-dosechemotherapy with or without radiation and allogenic or autologous bonemarrow transplantation can be employed. The high-dose chemotherapy mayemploy the original drug(s) or be altered to include additional agents.The development of new drugs, non-cross resistant with mdr phenotypes,is required to further the curative potential of current regimens and tofacilitate curative interventions in previously treated patients.

The in vitro anti-tumor activity of a novel class of natural productscalled illudins has been examined by Kelner, M. et al., Cancer Res., 47,3186 (1987). Illudin M was purified and submitted for evaluation to theNational Cancer Institute Division of Cancer Treatment (NCI DCT) in vivodrug screening program. Illudin M significantly increased the life spanof rats with Dunning leukemia, but had a low therapeutic index in solidtumor systems. The extreme toxicity of illudins has prevented anyapplications in human tumor therapy. Recently, synthetic analogs of theilludins have been developed which exhibit promising antitumor activity,including those analogs disclosed in U.S. Pat. Nos. 5,439,936 and5,523,490.

However, despite these developments, there exists a continuing need forchemotherapeutic agents which inhibit tumor growth, especially solidtumor growth, and which have an adequate therapeutic index to beeffective for in vivo treatment.

SUMMARY OF THE INVENTION

The invention provides a compound of formula I:

wherein

R₁ is hydrogen, hydroxy, mercapto, amino, halo, carboxy, nitro, or—(CH₂)_(n)—(X)—(Y);

n is 0 to 4;

X is oxy (—O—), thio (—S—), —N(R_(a))—, or absent;

Y is (C₃-C₆)cycloalkyl, aryl, heteroaryl, a saccharide, an amino acid, apeptide, or a 1 to 15 membered branched or unbranched carbon chainoptionally comprising 1, 2, or 3 non-peroxide oxy, thio, or —N(R_(a))—;wherein said chain may optionally be substituted on carbon with 1, 2, or3, oxo (═O), hydroxy, carboxy, halo, mercapto, nitro, —N(R_(b))(R_(c)),(C₃-C₆)cycloalkyl, aryl, heteroaryl, saccharides, amino acids, orpeptides; and wherein said chain may optionally be saturated orunsaturated;

R₂ is carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, halo(C₁-C₆)alkyl,—C(═O)NR_(d)R_(c), a saccharide, an amino acid, a peptide, or(C₁-C₆)alkyl substituted by 1 or 2 hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides, saccharides, or—C(═O)NR_(d)R_(e);

R₃ is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkylthio, aryl,heteroaryl, aryloxy, or heteroaryloxy;

R₄ is hydrogen or (C₁-C₆)alkyl; and R₅ is hydroxy, (C₁-C₆)alkoxy, or(C₁-C₆)alkanoyloxy; or R₄ and R₅ taken together are ethylenedioxy;

R₆ is hydrogen, carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,halo(C₁-C₆)alkyl, —C(═O)NR_(f)R_(g), a saccharide, an amino acid, apeptide, or (C₁-C₆)alkyl optionally substituted by 1 or 2 hydroxy,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides,saccharides, or —C(═O)NR_(f)R_(g);

R_(a) is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; and

R_(b), R_(c), R_(d), R_(e), R_(f) and R_(g) are each independentlyhydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; or R_(b) andR_(c), R_(d) and R_(e), or R_(f) and R_(g), together with the nitrogento which they are attached, are pyrrolidino, piperidino, or morpholino;

wherein any aryl, heteroaryl, aryloxy, or heteroaryloxy of Y, or R₃ mayoptionally be substituted by 1, 2, or 3 (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, (C₁-₆)alkoxycarbonyl,hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto,nitro, or —N(R_(h))(R_(j)); wherein each R_(h) and R_(j) isindependently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl;or R_(h) and R_(j) together with the nitrogen to which they are attachedare pyrrolidino, piperidino, or morpholino;

or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of formula I wherein: R₁ is—(CH₂)_(n)—(X)—(Y); n is 0 to 4; X is oxy, thio, —N(R_(a))—, or absent;Y is a monoprotected amino acid, a diprotected amino acid, a peptide, ora 1 to 15 membered branched or unbranched carbon chain optionallycomprising 1, 2, or 3 non-peroxide oxy, thio, or —N(R_(a))—; whereinsaid chain is substituted with 1, 2, or 3 peptides; and wherein saidchain may optionally be saturated or unsaturated; R₂ is hydrogen or(C₁-C₆)alkyl; R₃ is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkylthio, aryl, heteroaryl, aryloxy, or heteroaryloxy; R₄ ishydrogen or (C₁-C₆)alkyl; and R₅ is hydroxy, (C₁-C₆)alkoxy, or(C₁-C₆)alkanoyloxy; or R₄ and R₅ taken together are ethylenedioxy; R₆ ishydrogen, carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,halo(C₁-C₆)alkyl, —C(═O)NR_(f)R_(g), a saccharide, an amino acid, apeptide, or (C₁-C₆)alkyl optionally substituted by 1 or 2 hydroxy,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides,saccharides, or —C(═O)NR_(f)R_(g); R_(a) is hydrogen, (C₁-C₆)alkyl,(C₁-C₆)alkanoyl, phenyl or benzyl; and R_(b), R_(c), R_(d), R_(e), R_(f)and R_(g) are each independently hydrogen, (C₁-C₆)alkyl,(C₁-C₆)alkanoyl, phenyl or benzyl; or R_(b) and R_(c), R_(d) and R_(e),or R_(f) and R_(g), together with the nitrogen to which they areattached, are pyrrolidino, piperidino, or morpholino; wherein any aryl,heteroaryl, aryloxy, or heteroaryloxy of Y, or R₃ may optionally besubstituted by 1, 2, or 3 (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl, hydroxy(C₁-C₆)alkyl,halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto, nitro, or—N(R_(h))(R_(j)); wherein each R_(h) and R_(j) is independentlyhydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; or R_(h) andR_(j) together with the nitrogen to which they are attached arepyrrolidino, piperidino, or morpholino; or a pharmaceutically acceptablesalt thereof Preferably, Y is (C₁-C₆)alkyl substituted with a peptide.

The invention also provides a compound of formula I wherein:

R₁ is hydrogen, hydroxy, mercapto, amino, halo, carboxy, nitro, or—(CH₂)_(n)—(X)—(Y); n is 0 to 4; X is oxy, thio, —N(R_(a))—, or absent;Y is (C₃-C₆)cycloalkyl, aryl, heteroaryl, a saccharide, an amino acid, apeptide, or a 1 to 15 membered branched or unbranched carbon chainoptionally comprising 1, 2, or 3 non-peroxide oxy, thio, or —N(R_(a))—;wherein said chain may optionally be substituted on carbon with 1, 2, or3, oxo, hydroxy, carboxy, halo, mercapto, nitro, —N(R_(a))(R_(c)),(C₃-C₆)cycloalkyl, aryl, heteroaryl, saccharides, amino acids, orpeptides; and wherein said chain may optionally be saturated orunsaturated; R₂ is hydrogen or (C₁-C₆)alkyl; R₃ is hydrogen,(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkylthio, aryl, heteroaryl,aryloxy, or heteroaryloxy; R₄ is hydrogen or (C₁-C₆)alkyl; and R₅ ishydroxy, (C₁-C₆)alkoxy, or (C₁-C₆)alkanoyloxy; or R₄ and R₅ takentogether are ethylenedioxy; R₆ is carboxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, —C(═O)NR_(f)R_(g), a saccharide, an amino acid, apeptide, or (C₁-C₆)alkyl substituted by 1 or 2 (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides, saccharides, or—C(═O)NR_(f)R_(g); R_(a) is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,phenyl or benzyl; and R_(b), R_(c), R_(d), R_(c), R_(f) and R_(g) areeach independently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl orbenzyl; or R_(b) and R_(c), R_(d) and R_(e), or R_(f) and R_(g),together with the nitrogen to which they are attached, are pyrrolidino,piperidino, or morpholino; wherein any aryl, heteroaryl, aryloxy, orheteroaryloxy of Y, or R₃ may optionally be substituted by 1, 2, or 3(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy,halo, carboxy, mercapto, nitro, or —N(R_(h))(R_(j)); wherein each R_(h)and R_(j) is independently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,phenyl or benzyl; or R_(h) and R_(j) together with the nitrogen to whichthey are attached are pyrrolidino, piperidino, or morpholino; or apharmaceutically acceptable salt thereof.

The invention also provides dimeric compounds comprising two compoundsof formula (I), connected by a linker. The linker can be, for example,an alkyl or ester based linker group. Examples of suitable linkersinclude —(CH₂)_(p)—O—(CH₂)_(q)—, —(CH₂)_(r)—, and—CH₂—S—CH₂C(O)—O—(CH₂)₂—O—C(O)CH₂—S—CH₂—; wherein p and q are eachindividually an integer from 1 to 8, inclusive; and r is an integer from1 to 16, inclusive. Preferably, r is an integer from 1 to 8, inclusive.As would be apparent to one skilled in the art, other linkers ofapproximately the same length can also be used. Two compounds of formulaI can conveniently be linked, for example, by replacing R₁, R₃, R₄ orR₅, independently, on each compound of formula I, with the bifunctionallinker. When the linkage is through R₁, the linker is preferably—CH₂—O—CH₂— or —CH₂—S—CH₂C(O)—O—(CH₂)₂—O—C(O)CH₂—S—CH₂—.

The invention also provides compounds of formula I wherein R₁. is—(CH₂)_(n)—(X)—(Y); n is 1 to 4 (preferably n is 1); X is oxy, thio, or—N(R_(a))—(preferably X is thio); Y is a 2 to 15 membered branched orunbranched carbon chain optionally comprising 1, 2, or 3 non-peroxideoxy, thio, or —N(R_(a))—; wherein said chain is substituted on carbonwith 1, 2, or 3, oxo, carboxy, mercapto, —N(R_(b))(R_(c)),(C₃-C₆)cycloalkyl, aryl, heteroaryl, saccharides, amino acids, orpeptides; and wherein said chain may optionally be saturated orunsaturated (preferably Y is a 2 to 6 membered branched or unbranchedcarbon chain that is substituted on carbon with 1, 2, or 3, oxo,heteroaryl, amino acids, or peptides); R₂ is hydrogen or (C₁-C₆)alkyl;R₃ is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkylthio, aryl,heteroaryl, aryloxy, or heteroaryloxy; R₄ is hydrogen or (C₁-C₆)alkyl;and R, is hydroxy, (C₁-C₆)alkoxy, or (C₁-C₆)alkanoyloxy; or R₄ and R₅taken together are ethylenedioxy; R₆ is hydrogen, carboxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, halo(C₁-C₆)alkyl,—C(═O)NR_(f)R_(g), a saccharide, an amino acid, a peptide, or(C₁-C₆)alkyl optionally substituted by 1 or 2 hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides, saccharides, or—C(═O)NR_(f)R_(g); R_(a) is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,phenyl or benzyl; and R_(b), R_(c), R_(f) and R_(g) are eachindependently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl;or R_(b) and R_(c), or R_(f) and R_(g), together with the nitrogen towhich they are attached, are pyrrolidino, piperidino, or morpholino;wherein any aryl, heteroaryl, aryloxy, or heteroaryloxy of Y, or R₃ mayoptionally be substituted by 1, 2, or 3 (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl,hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto,nitro, or —N(R_(h))(R_(j)); wherein each R_(h) and R_(j) isindependently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl;or R_(h) and R_(j) together with the nitrogen to which they are attachedare pyrrolidino, piperidino, or morpholino; or a pharmaceuticallyacceptable salt thereof.

The invention also provides a compound of formula II:

wherein

R₂ is (C₁-C₆)alkyl;

R₃ is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkylthio, aryl,heteroaryl, aryloxy, or heteroaryloxy;

R₄ is hydrogen or (C₁-C₆)alkyl; and R₅ is hydroxy, (C₁-C₆)alkoxy, or(C₁-C₆)alkanoyloxy; or R₄ and R₅ taken together are ethylenedioxy;

R₆ is hydrogen, carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,halo(C₁-C₆)alkyl, —C(═O)NR_(f)R_(g), a saccharide, an amino acid, apeptide, or (C₁-C₆)alkyl optionally substituted by 1 or 2 hydroxy,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides,saccharides, or —C(═O)NR_(f)R_(g);

R₇ is carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, halo(C₁-C₆)alkyl,—C(═O)NR_(d)R_(e), a saccharide, an amino acid, a peptide, or(C₁-C₆)alkyl substituted by 1 or 2 hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides, saccharides, or—C(═O)NR_(d)R_(e);

R_(d), R_(e), R_(f) and R_(g) are each independently hydrogen,(C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; or R_(d) and R_(e), orR_(f) and R_(g), together with the nitrogen to which they are attached,are pyrrolidino, piperidino, or morpholino;

wherein any aryl, heteroaryl, aryloxy, or heteroaryloxy of R₃ mayoptionally be substituted by 1, 2, or 3 (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl,hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto,nitro, or —N(R_(h))(R_(j)); wherein each R_(h) and R_(j) isindependently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl;or R_(h) and R_(j) together with the nitrogen to which they are attachedare pyrrolidino, piperidino, or morpholino;

or a pharmaceutically acceptable salt thereof.

Compounds of the invention are useful as antineoplastic agents, i.e., toinhibit tumor cell growth in vitro or in vivo, in mammalian hosts, suchas humans or domestic animals, and are particularly effective againstsolid tumors and multi-drug resistant tumors. Thus, the inventionprovides a method comprising inhibiting cancer cells, by contacting saidcells, in vitro or in vivo, with an effective amount of a compound ofthe invention. The invention also provides a therapeutic methodcomprising treating cancer (i.e., inhibiting tumor cell growth) byadministering a compound of the invention to a mammal (e.g. a human) inneed of such therapy.

The present compounds may be targeted to a particular tumor by attachingthe compound to a reagent which is capable of binding to atumor-associated antigen. The antigen may be located on a tumor or inthe tumor cell area. Suitable reagents include polyclonal and monoclonalantibodies. The compound-reagent complex or conjugate may furthercomprise a linker (e.g. a linker as described hereinabove) for attachingthe compound to the reagent. Accordingly, the invention also provides acompound comprising a compound of formula I or formula II and a reagent(e.g. a polyclonal or monoclonal antibody) which is capable of bindingto a tumor-associated antigen.

The present invention also provides a pharmaceutical composition (e.g. apharmaceutical unit dosage form), comprising one or more compounds ofthe invention in combination with a pharmaceutically acceptable diluentor carrier.

The invention also provides a compound of the invention (e.g. a compoundof formula I or II, a dimer thereof, or a conjugate comprising acompound of formula I or II and a reagent that is capable of binding toa tumor-associated antigen, or a salt thereof) for use in medicaltherapy (preferably for use in treating cancer, e.g. solid tumors), aswell as the use of a compound of the invention for the manufacture of amedicament useful for the treatment of cancer, e.g. solid tumors.

The invention also provides processes and novel intermediates disclosedherein that are useful for preparing compounds of the invention. Some ofthe compounds of the invention are useful to prepare other compounds ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows representative compounds of the invention (compounds 1 to9) and intermediate compounds 10 or 11.

FIG. 2 shows representative compounds of the invention (compounds 13 to16).

FIG. 3 shows representative compounds of the invention (compounds 17 and18).

DETAILED DESCRIPTION

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, etc. denote bothstraight and branched groups; but reference to an individual radicalsuch as “propyl” embraces only the straight chain radical, a branchedchain isomer such as “isopropyl” being specifically referred to. Aryldenotes a phenyl radical or an ortho-fused bicyclic carbocyclic radicalhaving about nine to ten ring atoms in which at least one ring isaromatic. Heteroaryl encompasses a radical attached via a ring carbon ofa monocyclic aromatic ring containing five or six ring atoms consistingof carbon and one to four non-peroxide oxygen, sulfur, or N(R_(y))wherein R_(y) is absent or is H, O, (C₁-C₄)alkyl, phenyl or benzyl, aswell as a radical of an ortho-fused bicyclic heterocycle of about eightto ten ring atoms derived therefrom, particularly a benz-derivative orone derived by fusing a propylene, trimethylene, or tetramethylenediradical thereto.

The term “inhibit” or “inhibiting” means decreasing tumor cell growthrate from the rate which would occur without treatment, and/or causingtumor mass to decrease. Inhibiting also includes causing a completeregression of the tumor. Thus, the present analogs can either becytostatic or cytotoxic to tumor cells.

The method of the invention can be practiced on any mammal having asusceptible cancer, i.e., a malignant cell population or tumor.Compounds of the invention are effective on human tumors in vivo as wellas on human tumor cell lines in vitro. The present compounds may beparticularly useful for the treatment of solid tumors for whichrelatively few treatments are available. Such tumors include epidermoidand myeloid tumors, acute (AML) or chronic (CML). Such tumors alsoinclude, nonsmall cell, squamous, liver, cervical, renal, adrenal,stomach, esophageal, oral and mucosal tumors, as well as lung, ovarian,breast and colon carcinoma, and melanomas (including amelanoticsubtypes). The present compounds can also be used against endometrialtumors, bladder cancer, pancreatic cancer, lymphoma, Hodgkin's disease,prostate cancer, sarcomas and testicular cancer as well as againsttumors of the central nervous system, such as brain tumors,neuroblastomas and hematopoietic cell cancers such as B-cellleukemia/lymphomas, myelomas, T-cell leukemia/lymphomas, small cellleukemia/lymphomas, as well as null cell, sezary, monocytic,myelomonocytic and Hairy cell leukemia. These leukemia/lymphomas can beeither acute (ALL) or chronic (CLL).

The term “saccharide” includes monosaccharides, disaccharides,trisaccharides and polysaccharides. The term includes glucose, sucrosefructose and ribose, as well as deoxy sugars such as deoxyribose and thelike. Saccharide derivatives can conveniently be prepared as describedin International Patent Applications Publication Numbers WO 96/34005 and97/03995. A saccharide an conveniently be linked to the remainder of acompound of formula I or II through an ether bond.

The term “amino acid,” comprises the residues of the natural amino acids(e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, eu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as wellas unnatural amino acids (e.g. phosphoserine, phosphothreonine,phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, citruline, α-methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). Theterm also comprises natural and unnatural amino acids bearing aconventional amino protecting group (e.g. acetyl or benzyloxycarbonyl),as well as natural and unnatural amino acids protected at the carboxyterminus (e.g. as a (C₁-C₆)alkyl, phenyl or benzyl ester or amide; or asan a-methylbenzyl amide). Other suitable amino and carboxy protectinggroups are known to those skilled in the art (See for example, T. W.Greene, Protecting Groups In Organic Synthesis; Wiley: New York, 1981,and references cited therein). An amino acid can be linked to theremainder of a compound of formula I through the carboxy terminus, theamino terminus, or through any other convenient point of attachment,such as, for example, through the sulfur of cysteine.

The term “peptide” describes a sequence of 2 to 25 amino acids (e.g. asdefined hereinabove) or peptidyl residues. The sequence may be linear orcyclic. For example, a cyclic peptide can be prepared or may result fromthe formation of disulfide bridges between two cysteine residues in asequence. A peptide can be linked to the remainder of a compound offormula I through the carboxy terminus, the amino terminus, or throughany other convenient point of attachment, such as, for example, throughthe sulfur of a cysteine. Preferably a peptide comprises 3 to 25, or 5to 21 amino acids. Peptide derivatives can be prepared as disclosed inU.S. Pat. Nos. 4,612,302; 4,853,371; and 4,684,620, or as described inthe Examples hereinbelow. Peptide sequences specifically recited hereinare written with the amino terminus on the left and the carboxy terminuson the right.

It has been shown that certain peptides specifically bind to specifictumor-associated antigens in a manner analogous to the binding ofantibodies to such antigens. See Arap et al. Science, 1998, 279, 5349,377-380. Thus, pharmaceutical agents which comprise a peptide that iscapable of binding to specific receptors on tumor cells, can bedelivered preferentially to such tumor cells. As a result, one preferredembodiment of the invention provides a compound of formula I or IIcomprising a peptide capable of specifically binding to atumor-associated antigen. Preferred peptides include-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ ID NO:1), -Cys-Asp-Gly-Arg-Cys(SEQ ID NO:2), and-Cys-Asp-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQ IDNO:3).

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine anti-tumor activity using Test Aor Test B, described hereinbelow, or using other tests which are wellknown in the art. In preferred compounds of formula I, the absolutestereochemistry at the carbon bearing R₄ and R₅ is (R).

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituentsSpecifically, (C₁-C₄)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, or sec-butyl; (C₁-C₆)alkyl can be methyl, ethyl,propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, orhexyl; (C₁-C₈)alkyl can be methyl, ethyl, propyl, isopropyl, butyl,iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, septyl, or octyl;(C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl; (C₁-C₄)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy,butoxy, iso-butoxy, or sec-butoxy; (C₁-C₆)alkoxy can be methoxy, ethoxy,propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy,or hexyloxy; (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 1-pentenyl,2-pentenyl, 3-pentenyl, 4-pentenyl, 3-methyl-2-butenyl,3-methyl-3-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,5-hexenyl, or 4-methyl-3-pentenyl; (C₁-C₆)alkanoyl can be acetyl,propanoyl or butanoyl; halo(C₁-C₆)alkyl can be iodomethyl, bromomethyl,chloromethyl, fluoromethyl, trichloromethyl, trifluoromethyl,2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl;hydroxy(C₁-C₆)alkyl can be hydroxymethyl, 1-hydroxyethyl,2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl,1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl,1-hydroxyhexyl, or 6-hydroxyhexyl; (C₁-C₆)alkoxycarbonyl can bemethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C₁-C₆)alkylthiocan be methylthio, ethylthio, propylthio, isopropylthio, butylthio,isobutylthio, pentylthio, or hexylthio; (C₁-C₆)alkanoyloxy can befornyloxy, acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy,pentanoyloxy, or hexanoyloxy; aryl can be phenyl, indenyl, or naphthyl;and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl,isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl,tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or itsN-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or itsN-oxide).

A specific value for R, is hydroxy, halo, carboxy, aryl, heteroaryl, asaccharide, an amino acid, or a peptide.

Another specific value for R₁ is —(CH₂)_(n)—(X)—(Y); wherein n is 0 to4; X is oxy, thio, —N(R_(a))—, or absent; and Y is a peptide, or(C₁-C₆)alkyl substituted with a peptide.

Another specific value for R₁ is hydrogen or (C₁-C₆)alkyl, optionallysubstituted with 1 or 2 hydroxy, halo, methoxy or ethoxy.

Another specific value for R₁ is —(CH₂)_(n)—(X)—(Y); X is oxy, thio, or—N(R_(a))—; and Y is CH₂OC(O)(C₁-C₄)alkyl, CH₂C(O)—O—(CH₂)₂—O—C(O)CH₂SH,(CH₂)₂—O—(CH₂)₂halo, (C₁-C₄)alkyl-O—(C₁—C₄)alkyl, CH₂CO₂(C₁-C₄)alkyl,CH₂CO₂H, aryl(C₁-C₄)alkyl, a saccharide, an amino acid, or (C₁-C₈)alkyloptionally substituted with 1 or 2 hydroxy or halo; wherein any aryl orheteroaryl of Y may optionally be substituted with 1 or 2 hydroxy, halo,(C₁-C₄)alkyl or (C₁-C₄)alkoxy.

Another specific value for R₁ is —CH₂—(X)—(Y), wherein X is oxy, thio,or —N(R_(a))—; and Y is (C₁-C₈)alkyl optionally substituted with 1 or 2hydroxy, halo, carboxy, oxo, mercapto, —N(R_(b))(R_(c)),(C₃-C₆)cycloalkyl, aryl, heteroaryl, saccharides, amino acids, orpeptides; wherein any aryl or heteroaryl of Y may optionally besubstituted by 1, 2, or 3(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl, hydroxy(C₁-C₆)alkyl,halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto, nitro, or—N(R_(h))(R_(j)); wherein each R_(h) and R_(j) is independentlyhydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; or R_(h) andR_(j) together with the nitrogen to which they are attached form apyrrolidino, piperidino, or morpholino radical.

Another specific value for R₁ is —(CH₂)_(n)—(X)—(Y); n is 1 or 2; X isoxy, thio, or —N(R_(j))—; and Y is (C₁-C₆)alkyl or (C₂-C₆)alkenyl,optionally substituted with 1 or 2 oxo, hydroxy, carboxy, halo,mercapto, nitro, —N(R_(f))(R_(c)), (C₃-C₆)cycloalkyl, aryl, heteroaryl,saccharides, amino acids, or peptides; wherein any aryl or heteroaryl ofY may optionally be substituted by 1, 2, or 3 (C₁-C₆)alkyl, (C₁₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl,hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto,nitro, and —N(R_(h))(R_(j)); wherein each R_(h) and R₁ is independentlyhydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl and benzyl; or R_(h) andR₁ together with the nitrogen to which they are attached form apyrrolidino, piperidino, or morpholino radical.

Another specific value for R₁ is —CH₂-[sulfur-linked -cysteine]-R_(x)wherein R_(x) is an amino acid or a peptide comprising 2 to 24 aminoacids.

Another specific value for R₁ is —CH₂-[sulfur-linked—N-acylcysteine]-R_(x) wherein R_(x) is an amino acid or a peptidecomprising 2 to 24 amino acids.

Another specific value for R₁ is —CH₂-[sulfur-linked-glutathione].

Another specific value for R₁ is2-[(R)-α-methylbenzyl-aminocarbonyl]-2-(acylamino)ethylthiomethyl.Preferably, the 2-position of the ethyl group has the (S) configurationof cysteine.

A more specific value for R₁ is hydrogen, methyl, hydroxymethyl,methoxymethyl, or acetoxymethyl.

A specific value for R₂ is hydroxymethyl, methoxymethyl, oracetoxymethyl.

Another specific value for R₂ is carboxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, or —C(═O)NR_(d)R_(e).

Another specific value for R₂ is (C₁-C₆)alkyl substituted by 1 or 2hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids,peptides, saccharides, or —C(═O)NR_(d)R_(e).

Another specific value for R₂ is —CH₂-[sulfur-linked -cysteine]-R_(x)wherein R_(x) is an amino acid or a peptide comprising 2 to 24 aminoacids.

Another specific value for R₂ is—CH₂-[sulfur-linked-N-acylcysteine]-R_(x) wherein R_(x) is an amino acidor a peptide comprising 2 to 24 amino acids.

Another specific value for R₂ is —CH₂-[sulfur-linked-glutathione].

A specific value for R₃ is hydrogen.

A specific value for R₄ is methyl.

A specific value for R₅ is hydroxy.

A specific value for R₆ is hydrogen.

A specific value for R₆ is carboxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, or —C(═O)NR_(f)R_(g).

A specific value for R₆ is (C₁-C₆)alkyl optionally substituted by 1 or 2hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids,peptides, saccharides, or —C(═O)NR_(f)R_(g).

Another specific value for R₆ is —CH₂-[sulfur-linked-cysteine]-R_(x)wherein R_(x) is an amino acid or a peptide comprising 2 to 24 aminoacids.

Another specific value for R₆ is—CH₂-[sulfur-linked-N-acylcysteine]-R_(x) wherein R_(x) is an amino acidor a peptide comprising 2 to 24 amino acids.

A more specific value for R₆ is methyl or hydroxymethyl.

A specific value for R₇ is (C₁-C₆)alkyl substituted by 1 or 2 hydroxy,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides,saccharides, or —C(═O)NR_(d)R_(e).

A specific value for R₇ is hydroxymethyl.

Specifically, R_(x) can be a peptide comprising 4 to 20 amino acids.

Another specific value for R_(x) is -Leu-Gly-Phe, -Phe-Leu-Gly,-Leu-Leu-Phe, -Gly-Phe, or -Leu.

Another specific value for R is -Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ IDNO:4), -Asp-Gly-Arg-Cys (SEQ ID NO:5), or-Asp-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQ ID NO:5).

A specific compound of the invention is a compound of formula I whereinR₂ is (C₁-C₆)alkyl, substituted with hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy, or carboxy; R₃ is H, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkylthio, aryl, heteroaryl, aryloxy, or heteroaryloxy; R₄ ishydrogen or (C₁-C₆)alkyl; R₅ is hydroxy; and R₆ is (C₁-C₆)alkyl,optionally substituted with hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy,or carboxy; or a pharmaceutically acceptable salt thereof.

Another specific compound of the invention is a compound of formula Iwherein R₁ is hydrogen or (C₁-C₆)alkyl, optionally substituted withhydroxy, halo, methoxy, ethoxy, or acetoxy; R₂ is hydroxymethyl,methoxymethyl, or acetoxymethyl; R₃ is hydrogen; R₄is methyl; R₅ ishydroxy; and R₆ is methyl or hydroxymethyl; or a pharmaceuticallyacceptable salt thereof.

Another specific compound of the invention is a compound of formula Iwherein R₁ is —(CH₂)_(m)—(X)—(Y); n is 0 to 4; X is oxy, thio,—N(R_(a))—, or absent; Y is a monoprotected amino acid or a diprotectedamino acid; and R₂ is hydrogen or (C₁-C₆)alkyl; or a pharmaceuticallyacceptable salt thereof.

Another specific compound of the invention is a compound of formula Iwherein R₁ is —CH₂-[sulfur-linked-N-acylcysteine],(S)-2-[(R)-α-methylbenzylaminocarbonyl]-2—(acylamino)ethylthiomethyl, or(R)-2-[(R)-α-methylbenzylaminocarbonyl]-2—(acylamino)ethylthiomethyl;and R₂ is hydrogen or (C₁-C₆)alkyl; or a pharmaceutically acceptablesalt thereof.

A specific compound of formula I is a compound which is:

or a pharmaceutically acceptable salt thereof.

A specific compound of formula II is a compound which is

or a pharmaceutically acceptable salt thereof.

A preferred compound is a compound of formula I wherein R₄ is hydrogenor (C₁-C₆)alkyl; and R₅ is hydroxy or acetoxy; wherein the absolutestereochemistry at the carbon bearing R₄ and R₅ is (R); or apharmaceutically acceptable salt thereof.

Processes for preparing compounds of the invention are provided asfurther embodiments of the invention and are illustrated by thefollowing procedures in which the meanings of the generic radicals areas given above unless otherwise qualified.

The compounds of the present invention (compounds of formula I and IIand salts thereof) may be derived from illudin S, hydroxymethylacylfulvene (HMAF, i.e., a compound of formula (I) wherein R₁ is CH₂OH,R₂ is CH₃, R₃ is hydrogen, R₄ is CH₃, R₅ is OH and R₆ is CH₃) andfulvene (i.e., a compound of formula (I) wherein R₁ is H, R₂ is CH₃, R₃is H, R₄ is CH₃, R₅ is OH and R₆ is CH₃) the syntheses of which areknown in the art (see e.g., WO 91/04754; WO 94/18151).

A compound of formula I wherein R₂ is hydroxymethyl can be prepared byoxidation of a corresponding compound of formula I wherein R₂ is methyl.The oxidation can conveniently be carried out using selenium dioxide andtert-butyl hydroperoxide under conditions similar to those described inExample 1.

A compound of formula I wherein R₂ is hydroxymethyl can be prepared froma corresponding compound of formula I wherein R₁ is hydrogen bytreatment with paraformaldehyde and sulfuric acid. The reaction canconveniently be carried out under conditions similar to those describedin Example 2.

A compound of formula I wherein R₂ is acetoxymethyl can be prepared byacylation of a corresponding compound of formula I wherein R₂ ishydroxymethyl. The acylation can conveniently be carried out usingacetic anhydride, under conditions similar to those described in Example3.

A compound of formula I wherein R₂ is methoxymethyl can be prepared byreacting a corresponding compound of formula I wherein R₂ ishydroxymethyl with methyl iodide and silver oxide. The reaction canconveniently be carried out under conditions similar to those describedin Example 7.

A compound of formula I wherein R₁ is methoxymethyl can be prepared byreacting a corresponding compound of formula I wherein R₁ ishydroxymethyl with methanol and sulfuric acid. The reaction canconveniently be carried out under conditions similar to those describedin Example 8, sub-part a.

A compound of formula I wherein R₁ is —CH₂-[sulfur-linked-cysteine] canbe prepared by reacting a corresponding compound of formula I wherein R₁is hydroxymethyl by coupling with cysteine. The reaction canconveniently be carried out under conditions similar to those describedin Example 10.

A compound of formula I wherein R₁ is—CH₂-[sulfur-linked-N-acylcysteine] can be prepared by reacting acorresponding compound of formula I wherein R₁ is hydroxymethyl bycoupling with N-acylcysteine. The reaction can conveniently be carriedout under conditions similar to those described in Example 11.

A compound of formula I wherein Ris2-[(R)-αmethylbenzyl-aminocarbonyl]-2-(acylamino)ethylthiomethyl can beprepared by reacting a corresponding compound of formula I wherein Ris—CH₂-[sulfur-linked-N-acylcysteine] with α-methylbenzylamine. Thereaction can conveniently be carried out under conditions similar tothose described in Example 12.

A compound of formula I wherein R₁ is —CH₂-[sulfur-linked-glutathione]can be prepared by reacting a corresponding compound of formula Iwherein R₁ is hydroxymethyl with glutathione. The reaction canconveniently be carried out under conditions similar to those describedin Example 14.

A compound of formula I wherein R₁ is—CH₂-[sulfur-linked-cysteine]-R_(x), or—CH₂-[sulfur-linked-N-acylcysteine]-R_(x) can be prepared by reacting acorresponding compound of formula I wherein R₁ is—CH₂-[sulfur-linked-cysteine] or —CH₂-[sulfur-linked-N-acylcysteine]with the requisite amino acid or peptide (R_(x)). The reaction canconveniently be carried out under conditions similar to those describedin Example 15.

Compounds of formula II can conveniently be prepared from illudin Susing procedures similar to those described in Example 17.

Compounds of the invention can also be prepared using techniques andintermediates similar to those described by T. McMorris et al.Tetrahedron, 1997, 53, 44, 14579-14590; T. McMorris et al. J. Org. Chem.1997, 62, 3015-3018; T. McMorris et al. Chem. Comm. 1997, 315-316; andT. McMorris et al. Experentia 1996, 52, 75-80; and those disclosed inU.S. Pat. Nos. 5,439,942; 5,439,936; 5,523,490; 5,536,176; WO 91/04754;WO 94/18151 and WO 98/03458. Some compounds of formula I or II can beused to prepare other compounds of formula I or II.

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compounds as saltsmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds of the invention can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of dosage forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, a triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of the invention to the skin are known to the art;for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the invention can be determined bycorrelating their in yitr activity, and in vivo activity in animalmodels, such as murine or dog models as taught for illudin analogs suchas those of U.S. Pat. Nos. 5,439,936 and 5,523,490, to activity inhigher mammals, such as children and adult humans as taught, e.g., inBorch et al. (U.S. Pat. No. 4,938,949).

The therapeutically effective amount of analog necessarily varies withthe subject and the tumor to be treated. It has been found thatrelatively high doses of representative compounds of the invention canbe administered due to the decreased toxicity compared to illudin S andM. For example, the maximum tolerated dose of Illudin S is about 250ug/kg, whereas compound 2 can be chronically administered at 35 mg/kgwithout toxicity. A therapeutic amount between 30 to 112,000 μg per kgof body weight is especially effective for intravenous administrationwhile 300 to 112,000 μg per kg of body weight is effective ifadministered intraperitoneally. As one skilled in the art wouldrecognize, the amount can be varied depending on the method ofadministration. The amount of the compound, or an active salt orderivative thereof, required for use in treatment will vary not onlywith the particular salt selected but also with the route ofadministration, the nature of the condition being treated and the ageand condition of the patient and will be ultimately at the discretion ofthe attendant physician or clinician.

The compound can conveniently be administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about30 μM. This may be achieved, for example, by the intravenous injectionof a 0.05 to 5% solution of the active ingredient, optionally in saline,or orally administered as a bolus containing about 1-100 mg of theactive ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day.

The cytotoxic and anti-tumor properties of a compound of the inventioncan be determined using pharmacological models which are well known tothe art, or using Test A and Test B described below.

Test A. In Vitro Studies

To assess cytotoxic effects, various concentrations of compounds of theinvention were added to cultures of MV522 (human lung carcinoma cellline), HL60 (myeloid leukemia cells), and 8392 (B-cellleukemia/lymphoma) cells for 48 hours, then cell growth/viability wasdetermined by trypan blue exclusion. As an alternative to 48 hourcontinuous exposure studies, cells were plated in liquid culture in 96well plates, exposed to various concentrations of compounds of theinvention for 2 hours, pulsed with [³H]-thymidine for one to two hoursand harvested onto filter papers. The filter papers were added to vialscontaining scintillation fluid and residual radioactivity determined ina beta (scintillation) counter.

Data from Test A, for representative compounds of the invention, isshown in Table 1. Values are reported as mean±1 standard deviation;units are nanomoles per liter; and NT signifies not tested.

TABLE 1 2 hour IC₅₀ (nm/l) Compound MV522 HL60 8392 1 2,640 ± 360   NT37,000 ± 2,300  2 11,300 ± 1,500  NT NT 3 19,600 ± 9,700  15,600 ±4,600  62,000 ± 3,600  4 20,400 ± 6300   >40,000 NT 5 24,000 ± 6,100 38,400 ± 9,000  NT 6 NT NT NT 7 7,700 ± 3,500 6,000 ± 1,200 NT8 >80,000 >80,000 NT 9 >50,000 49,800 ± 20,000 NT 13 10,000 ± 1,800  NTNT 14 3,050 ± 550   NT NT 15 NT NT NT

Test B. In Vivo Studies

Several representative compounds of the invention were chosen for invivo studies. The anticancer agent mitomycin C was used as apharmaceutical control. Drug therapy was started 10 days afterinoculation on a daily basis via IP route for 5 consecutive days. Theanimals were monitored for 3 weeks after start of therapy. The MTD wasreached for the control agent mitomycin C but not for compounds 1 or 2.

BALB/c nu/nu 4-week old female mice weighing 18-22 g were obtained fromSimonsen, Inc. (Gilroy, Calif.) and maintained in the athymic mousecolony of the University of California (San Diego, Calif.) underpathogen free conditions using HEPA filter hoods. Animals were providedwith sterilized food and water ad libitum in groups of 5 in plasticcages vented with polyester fiber filter covers. Clean, sterilizedgowns, glove, face masks, and shoe and hood covers were worn by allpersonnel handling the animals. All studies were conducted in accordancewith guidelines of the NIH “Guide for Care and Use of Animals” andapproved by the University Institutional Animal Care and Use Committee(Protocol 3-006-2)

The MV522 lung carcinoma line used for xenograft studies was derived asdescribed by Kelner et al. (Anticancer Res., 15: 867-872; 873-878(1995)) and maintained in antibiotic-free RPMI 1640 (Mediatech, Herndon,Va.) supplemented with 10% fetal bovine serum and 2 mM glutamine in 37°C. humidified carbon dioxide incubator.

Mice were randomized into treatment groups of five animals each forinitial studies and groups of 16-20 animals for confirming analogueefficacy. Each animal was earmarked and followed individually throughoutthe experiments. Mice received s.c. injections of the parental cell lineMV522 using 10 million cells/inoculation over the shoulder. Ten daysafter s.c. implantation of the MV522 cells, when s.c. tumors wereapproximately 3×3 mm in size, animals received the desired drug anddosage. The effect of the compound on life span was calculated frommedian survival.

Although MV522 cells kill mice by metastases, primary s.c. tumor growthover the shoulder was monitored starting on the first day of treatmentand at weekly intervals thereafter. Tumor size was measured in twoperpendicular diameters. Tumor weights were estimated according to theformula w=(width)²×length/2). Relative weights (RW) were calculated tostandardized variability in tumor size among test groups at initiationof treatment sing the formula RW=Wt/wi, where Wi is the tumor weight fora given animal at beginning of drug treatment and Wt is tumor weight ata subsequent time. Animals were necropsied, and organs were examined forevidence of metastases.

Comparison of survival curves between groups of animals was by themethod of Kaplan and Meier. For comparison of relative tumor weightsbetween multiple groups of animals, ordinary ANOVA followed byTukey-Kramer multiple Comparison post ANOVA analysis was performed(Kelner et al. (Anticancer Res., 15: 867-872; 873-878 (1995)).Probability values (p) less than 0.05 were considered statisticallysignificant.

Data from Test B, for representative compounds of the invention, isshown in Table 2. Data was collected using eight animals in the controlstudies and four animals in each compound study. Values representrelative tumor weight or Wt/Wi (day ten by definition is 1.0). Drug wasadministered ip. on days 10, 11, 12, 13, and 14 (QD×5 days).

TABLE 2 Compound Day 17 Day 24 Day 31 control (saline) 4.3 ± 0.7 7.0 ±1.3 11.8 ± 3.0   1 (14 mg/kg) 1.4 ± 0.7 7.0 ± 1.3 11.8 ± 3.0   2 (35mg/kg) 2.1 ± 0.2 3.3 ± 0.3 4.0 ± 0.9 13 (32 mg/kg) 2.3 ± 0.5 4.1 ± 0.84.7 ± 1.5 14 (28 mg/kg) 3.2 ± 0.6 4.1 ± 0.8 6.0 ± 1.8

The data in Tables 1 and 2 shows that representative compounds of theinvention are potent cytotoxic and anti-tumor agents.

The invention will now be illustrated by the following non-limitingExamples, wherein, unless other wise stated: Melting points areuncorrected; ¹H and ¹³C NMR spectra were measured at 300 and 75 MHz;High resolution mass spectra were determined at the University ofMinnesota Mass. Spectrometry Service Laboratory; Chromatography wasperformed of silica gel (Davisil 230-425 mesh, Fisher Scientific), withethyl acetate:hexanes as the eluent; Analytical TLC was carried out onWhatman 4420 222 silica gel plates; and Reactions were routinelymonitored by TLC. Synthesis of illudin S, hydroxymethylacylfulvene(HMAF) and fulvene were prepared as previously described (see, e.g., WO91/04754; WO 94/18151).

EXAMPLES Example 1

Compound 1 (formula I wherein R₁ is hydrogen; R₂ is hydroxymethyl; R₃ ishydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ is methyl)

To a stirred solution of acylfulvene (6.9 g, 32 mmol) in 7.0 ml ofEtOAc, were added 99% Selenium dioxide (1.75 g, 15.8 mmol), and 90%tert-butyl hydroperoxide (6 ml, d 0.901, 60 mmol). The mixture wasstirred at room temperature for 24 hr and then partitioned between EtOAcand saturated sodium sulfite (3×3 ml), followed by saturated brine. Theorganic extract was then dried over MgSO₄. After concentration, thecrude product was chromatographed to give 922.5 mg of the title compound(12%) as a yellow-orange gum, and 5.8 g of Acylfulvene (84%); ¹H NMR(CDCl₃) δ0.76 (ddd, 1H), 1.14 (ddd, 1H), 1.27 (ddd, 1H), 1.36 (s, 3H),1.54 (ddd, 1H), 2.03 (s, 3H), 4.02 (s, 1 H), 4.56 (s, 2H), 6.67 (s, 1H),7.29 (s, 1H); ¹³C NMR (CDCl₃) δ197.9, 161.5, 146.0, 140.2, 132.9, 127.0,119.8, 76.6, 59.9, 37.5, 27.7, 17.0, 14.7, 9.9; MS m/e 232 (M⁺); UV λmax (EtOH) 327.2 nm (ε7631).

Example 2

Compound 2 (formula I wherein R₁ is hydroxymethyl; R₂ is hydroxymethyl;R₃ is hydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ is methyl)

Paraformaldehyde (36.3 g) was added to a solution of dilute H₂SO₄ (1.5M, 275 ml), and Me₂CO (300 ml). The mixture was stirred and heated todissolve all the solid. To the cooled (0° C.) solution compound 1(922.50 mg, 3.98 mmol) was added, the resulting solution was stirred andallowed to warm to room temperature. After 24 hr, the orange-yellowmixture was extracted with EtOAc (2×300 ml) and the combined extractswashed with saturated NaHCO₃ (30 ml), followed by saturated brine. Theorganic extract was then dried over MgSO₄. Removal of solvent andchromatography of the residue on Si gel with EtOAc-hexanes afforded357.3 mg (34%) of the title compound as a dark yellow-orange gum; ¹H NMR(CDCl₃) δ6 0.79 (ddd, 1H), 1.16 (ddd, 1H), 1.26 (ddd, 1H), 1.38 (s, 3H),1.41 (ddd, 1H), 2.20 (s, 3H), 3.90 (s, 1H), 4.63 (s, 2H), 4.74 (s, 2H),7.25 (s, 1H), MS m/e 262 (M⁺); UV λ max (EtOH) 330.0 nm (ε5011).

Example 3

Compound 3 (formula I wherein R₁ is hydrogen; R₂is acetoxymethyl; R₃ ishydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ is methyl)

To a stirred solution of compound 1 (76.0 mg, 0.328 mmol) in 2 ml ofacetic anhydride at room temperature, anhydrous sodium acetate (126.2mg, 1.54 mmol) was added. The mixture was stirred at room temperaturefor 24 hr, filtered to remove the NaOAc. The solution was placed underreduced pressure for 4 hr. The crude product was then partitionedbetween EtOAc and saturated NaHCO₃, followed by saturated brine, anddried over MgSO₄. After concentration the residue was chromatographed onSi gel with EtOAc-hexanes to give the title compound (50 mg, 56%) as ayellow gum; ¹H NMR (CDCl₃) δ0.77 (ddd, 1H), 1.16 (ddd, 1H), 1.34 (ddd,1H), 1.38 (s, 3H), 1.58 (ddd, 1H), 2.06 (s, 3H), (s 2.11 (s, 3H) 4.99(s, 2H), 6.69 (s, 1H), 7.27 (s, 1H); ¹³C NMR (CDCl₃) δ197.9, 170.8,163.6, 140.5, 140.0, 132.8, 126.7, 121.9, 76.7, 61.3, 37.9, 27.8, 20.9,17.3, 15.3, 10.3.

Example 4

Compound 4 (formula I wherein R₁ is acetoxymethyl; R₂ is hydroxymethyl;R₃ is hydrogen; R₄ is methyl; R is hydroxy; and R₆ is methyl)

To a stirred solution of compound 10 (430 mg, 1.49 mmol) in 3 ml ofEtOAc, 99% selenium dioxide (186 mg, 1.67 mmol) was added, followed by90% tert-butyl hydroperoxide (0.40 ml, d 0.901, 4.0 mmol). The mixturewas let stir at room temperature for 4 days, and then partitionedbetween EtOAc and saturated sodium sulfite (3×3 ml), followed bysaturated brine, and dried over magnesium sulfate. After concentrationthe crude product was chromatographed to give the title compound (8.5mg, 2%) as a yellow gum; ¹H NMR (CDCl₃) δ0.79 (ddd, 1H), 1.17 (ddd, 1H),1.39 (s, 3H), 1.43 (ddd, 1H), 1.52 (ddd, 1H), 2.09 (s, 3H), 2.14 (s,3H), 3.93 (s, 1H), 4.65 (q, 2H), 5.21 (q, 2H), 7.32 (s, 1H); MS m/e 304(M⁺); UV λ max (EtOH) 330.6 nm (ε5950).

The intermediate compound 10 was prepared as follows.

a. Compound 10. Anhydrous sodium acetate was added to a stirred solutionof HMAF (1.4 g, 5.7 mmol) in acetic anhydride (6 mL) at roomtemperature. After 18 hours, the mixture was filtered and the resultingsolution was placed under reduced pressure for 4 hours. The resultingmaterial was partitioned between ethyl acetate and saturated aqueoussodium bicarbonate, followed by brine, and the resulting ethyl acetatesolution was dried over magnesium sulfate. The solution was concentratedand purified by chromatography on silica gel with ethyl acetate:hexanesas the eluent to give compound 10 (1.4 g, 85%) as a yellow-orange gum;¹H NMR (CDCl₃) δ0.74 (ddd, 1H), 1.11 (ddd, 1H), 1.36 (s, 3H), 1.51 (ddd,1H), 2.04 (s, 3H), 2.09 (s, 3H), 2.17 (s, 3H), 3.90 (s, 1H), 5.10 (s,2h), 7.11 (s, 1H).

Example 5

Compound 5 (formula I wherein R₁ is acetoxymethyl; R₂ is acetoxymethyl;R₃ is hydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ is methyl)

To a stirred solution of compound 4 (2 mg, 0.0066 mmol) in 15 μl ofCH₂Cl₂ and 135 μl of acetic anhydride, anhydrous NaOAc (3.1 mg, 0.038mmol) was added. The reaction was let stir at room temperature for 4 hr,filtered to remove the NaOAc. The mixture was placed under reducedpressure to remove the acetic anhydride. The crude product was thenpartitioned between EtOAc and saturated NaHCO₃, followed by saturatedbrine, and dried over MgSO₄. After concentration the residue waschromatographed on Si gel with EtOAc-hexanes to give the title compound(0.9 mg, 40%) as a yellow gum. ¹H NMR (CDCl₃) δ0.79 (ddd, 1H), 1.15(ddd, 1H), 1.39 (s, 3H), 1.40 (ddd, 1H), 1.58 (ddd, 1H), 2.09 (s, 3H),2.093 (s, 3H), 2.10 (s, 3H), 3.89 (s, 1H), 5.08 (s, 2H), 5.17 (s, 2H),7.25 (s, 1H); MS m/e 346 (M⁺); UV λ max (EtOH) 332.1 nm (ε8378).

Example 6

Compound 6 (formula I wherein R₁ is hydroxymethyl; R₂ is acetoxymethyl;R₃ is hydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ is methyl)

To a stirred solution of compound 2 (76.0 mg, 0.290 mmol) in 1.5 ml ofacetic anhydride, anhydrous NaOAc (45.3 mg, 0.552 mmol) was added. Thereaction mixture was stirred at room temperature for 75 min then,filtered to remove the NaOAc. The mixture was partitioned between EtOAcand saturated NaHCO₃, followed by saturated brine, and dried over MgSO₄.The extract was concentrated under reduced pressure and the residue waschromatographed on Si gel with EtOAc-hexanes to give compound 4 (6.9 mg)as a yellow gum, compound 2 (10.0 mg) as a yellow orange gum, 3 minorproducts including compound 5, and the title compound (10.0 mg, 11.3%)as a yellow gum; ¹H NMR (CDCl₃) δ0.81 (ddd, 1H), 1.17 (ddd, 1H), 1.38(s, 3H), 1.60 (ddd, 1H), 2.06 (s, 3H), 2.24 (s, 3H), 3.89 (s, 1H), 4.74(m, 2H), 5.03 (m, 2H), 7.23 (1H); MS m/e 304 (M⁺); UV λ max (EtOH) 331.3nm (ε5921).

Example 7

Compound 7 (formula I wherein R₁ is hydrogen; R₂ is methoxymethyl; R₃ ishydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ is methyl)

To a stirred solution of compound 1 (300 mg, 1.29 mmol) in 10 ml ofCH₃CN, 1 ml of CH₃I, and Ag₂O (110 mg, 0.475 mmol) were added. Themixture was stirred at room temperature for 3 days, then filtered andconcentrated. The residue was chromatographed on Si gel withEtOAc-hexanes giving compound 1 (133 mg) and the title compound (53 mg,17%) as a dark orange gum; ¹NMR (CDCl₃) δ0.76 (ddd, 1H), 1.13 (ddd, 1H),1.34 (ddd, 1H), 1.38 (s, 3H), 2.05 (s, 3H), 3.41 (s, 3H), 3.96 (s, 1H),4.35 (s, 2H), 6.66 (s, 1H), 7.28 (s, 1H); ¹³C NMR (CDCl₃) δ197.8, 161.8,143.3, 140.2, 133.1, 126.7, 120.9, 76.6, 69.6, 58.3, 37.6, 27.8, 17.1,14.9, 10.0.

Example 8

Compound 8 (formula I wherein Ris methoxymethyl; R₂ is hydroxymethyl; R₃is hydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ is methyl)

To a stirred solution of compound 11 (830 mg, 3.19 mmol) in 10 ml ofEtOAc, 99% selenium dioxide (150 mg, 1.35 mmol) was added, followed by90% tert-butyl hydroperoxide (1 ml, d 0.901, 10.0 mmol). The reactionmixture was stirred at room temperature for 5 days, partitioned betweendiethyl ether and saturated sodium sulfite, followed by saturated brine,and dried over MgSO₄. After concentration the crude material waschromatographed to give the title compound. (47.2 mg, 5%) as a darkorange gum. 1H NMR (CDCl₃) δ0.74 (ddd, 1H), 1.08 (ddd, 1H), 1.32 (s,3H), 1.33 (ddd, 1H), 1.47 (ddd, 1H), 2.08 (s, 3H), 3.14 (br s, 1H), 3.35(s, 3H), 4.41 (q, 2H), 4.51 (br s, 2H), 7.28 (s, 1H).

a. Compound 11. The intermediate compound 11 (formula I wherein R₁ ismethoxymethyl; R₂is methyl; R₃ is hydrogen; R₄ is methyl; R₅ is hydroxy;and R₆ is methyl) was prepared as follows.

To a stirred solution of HMAF (320 mg, 1.3 mmol) in 3 ml Me₂CO, MeOH (3ml), and dilute H₂SO₄ (1M, 3 ml) were added. The reaction mixture wasstirred at room temperature for 24 hours and extracted with diethylether. The combined extracts were washed (saturated NaHCO₃, followed bybrine), and dried over MgSO₄. After concentration the residue waschromatographed on Si gel with EtOAc-hexanes affording compound 50 (290mg, 86%) as a dark orange gum; ¹H NMR (CDCl₃) δ0.62 (ddd, 1H), 0.98(ddd, 1H), 1.24 (ddd, 1H), 1.27 (s, 3H), 1.37 (ddd, 1H), 2.00 (s, 3H),2.04 (s, 3H), 3.26 (s, 3H), 3.91 (br s, 1H), 4.29 (q, 2H), 7.0 (s, 1H);¹³C NMR (CDCl₃) δ197.6, 159.7, 142.6, 138.8, 134.3, 129.7, 126.6, 75.9,65.3, 57.3, 37.3, 27.3, 15.7, 13.9, 12.9, 9.1.

Example 9

Compound 9 (formula I wherein R₁ is methoxymethyl; R₂ is methoxymethyl;R₃ is hydrogen; R₄is methyl; R₅ is hydroxy; and R₆ is methyl)

To a stirred solution of compound 8 (240 mg, 0.87 mmol) in 15 ml ofCH₃CN, 1.5 ml CH₃I, and Ag₂O (150 mg, 0.647 mmol) were added. Thereaction mixture was stirred at room temperature for 48 hr, filtered anconcentrated. The residue was chromatographed on Si gel withEtOAc-hexanes to give the title compound (25 mg, 10%) as a yellow-orangegum; ¹H NMR (CDCl₃) δ0.76 (ddd, 1H), 1.11 (ddd, 1H), 1.37 (s, 3H), 1.40(ddd, 1H), 1.53 (ddd, 1H), 2.13 (s, 3H), 3.36 (s, 3H), 3.38 (s, 3H),3.91 (s, 1H), 4.37 (q, 2H), 4.43 (q, 2H), 7.27 (s, 1H), ¹³C NMR (CDCl₃)δ198.0, 163.4, 142.8, 139.1, 132.6, 132.0, 127.2, 76.6, 67.2, 65.5,58.3, 57.8, 38.1, 27.5, 16.3, 14.8, 9.8.

Example 10

Compound 12 (formula I wherein R₁ is —CH₂-[sulfur-linked-cysteine]; R₂is methyl; R₃ is hydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ ismethyl)

To a solution of HMAF in acetone and 1M H₂SO₄ (1:1) was added oneequivalent of cysteine. The mixture was stirred at room temperatureovernight. A large amount of EtOAc was added and the water was removedby adding MgSO₄. Solid NaHCO₃ was also added in order to neutralize thesulfuric acid. The solution was filtered, concentrated, andchromatographed, to give compound 102 as a yellow gum: ¹H NMR (CDCl₃)δ0.78 (m, 1H), 0.89 (m, 1H), 1.06 (m, 1H), 1.31 (s, 3H), 1.43 (m, 1H),2.15 (s, 3H), 2.21 (s, 3H), 2 91-4.02 (m, 8H), 7.04 (s, 1H).

Example 11

Compound 13 (formula I wherein R₁ is—CH₂-[sulfur-linked-N-acylcysteine]; R₂ is methyl; R₃ is hydrogen; R₄ ismethyl; R₅ is hydroxy; and R₆ is methyl)

To a solution of HMAF (36 mg, 0.146 mmol) in 1:1 1M H₂SO₄/acetone (3 mL)was added N-acetyl cysteine (22.4 mg, 0.137 mmol) at room temperature.The mixture was stirred for 22 hours and was extracted with ethylacetate. The organic extracts were washed with saturated NaHCO₃ andsaline respectively. The solution was dried over MgSO₄. Afterconcentration, the crude product was chromatographed (adding 2-5% aceticacid to the normal solvent mixture, ethyl acetate and hexanes) to give45.5 mg of compound 103 (85% yield) as a yellow gum; ¹H NMR (CDCl₃)δ0.72 (m, 1H), 1.09 (m, 1H), 1.23 (m, 1H), 1.36 (s, 3H), 1.47 (m, 1H),2.07 (s, 3H), 2.10 (s, 3H), 2.13 (s, 3H), 2.97 (m, 1H), 3.14 (m, 1H),3.82 (dd, 3.82), 4.80 (m, 2H), 6.56 (d, J=7.2 Hz), 7.10 (s, 1H); MS m/e391 (M⁺), 373, 229, 185; HRMS for C₂₀H₂₅NO₅S calcd 391.1455, found391.1452.

Example 12

Compound 14 (formula I wherein R₁ is(S)-2-[(R)-α-methylbenzyl-aminocarbonyl]-2-(acylamino)ethylthiomethyl;R₂ is methyl; R₃ is hydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ ismethyl)

To a solution of compound 13 (40 mg, 0.102 mmol) in methylene chloride(1 mL) was added N-hydroxybenzotriazole (20 mg, 0.132 mmol),N,N-diisopropylcarbodiimide (20 μL, 0.12 mmol) and (d)-(+)-α-methylbenzylamine (12 μL, 0.093 mmol). The mixture was stirred for 1.5 hoursat room temperature. The mixture was partitioned between EtOAc andwater. The organic extract was dried over MgSO₄. After concentration thecrude product was chromatographed to give 33.6 mg of compound 14 (73%)as a yellow gum; ¹H NMR (CDCl₃) δ0.70 (m, 1H), 1.07 (m, 1H), 1.29 (m,1H), 1.35 (s, 3H), 1.48 (d, J=6.9 Hz, 3H), 1.51 (m, 1H), 1.93 (s, 3H),2.09 (s, 3H), 2.10 (s, 3H), 2.91 (m, 2H), 3.84 (dd, 2H), 4.61 (m, 1H),5.03 (m, 1H), 6.64 (d, J=7.8 Hz, 1H), 7.07 (s, 1H), 7.26 (m, 5H); ¹³CNMR (CDCl₃) δ197.5, 170.4, 169.2, 159.7, 142.6, 142.0, 138.3, 134.9,129.8, 128.7, 127.4, 126.4, 125.9, 77.4, 52.5, 49.3, 37.7, 34.4, 29.8,27.6, 23.1, 22.1, 16.3, 14.3, 13.1, 9.5; HRMS for C₂₈H₃₄N₂O₄S calcd494.2241, found 494.2225.

Example 13

Compound 15 (formula I wherein R₁ is(R)-2-[(R)-α-methylbenzyl-aminocarbonyl]-2-(acylamino)ethylthiomethyl;R₂ is methyl; R₃ is hydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ ismethyl)

The chromatography from Example 12 also provided 5.3 mg of compound 15(13%) as a yellow gum; ¹H NMR (CDCl₃) δ0.70 (m, 1H), 1.07 (m, 1H), 1.32(m, 1H), 1.34 (s, 3H), 1.45 (m, 1H), 1.48 (d, J=6.9 Hz, 3H), 2.03 (s,3H), 2.05 (s, 6H), 2.76 (m, 1H), 2.87 (m, 1H), 3.73 (dd, 2H), 4.50 (m,1H), 5.03 (m, 1H), 6.46 (d, J=7.5 Hz, 1H), 6.77 (d, J=7.8 Hz, 1H), 7.05(s, 1H), 7.31 (m, 5H); HRMS for C₂₈H₃₄N₂O₄S calcd 494.2241, found494.2238.

Example 14

Compound 16 (formula I wherein R₁ is —CH₂-[sulfur-linked-glutathione];R₂ is methyl; R₃ is hydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ ismethyl)

Using a procedure similar to that described in Example 10 exceptreplacing the cysteine used therein with glutathione, compound 16 wasprepared.

Example 15 General Procedure for the Synthesis of Compounds wherein R₁is —CH₂-[sulfur-linked-cysteine]-peptide,—CH₂-[sulfur-linked-cysteine]-(amino acid),—CH₂-[sulfur-linked-N-acylcysteine]-peptide, or—CH₂-[sulfar-linked-N-acylcysteine]-(amino acid)

Using solid phase peptide synthesis techniques, which are well known inthe art, the amino acids Leu, Phe and Gly were combined to give thefollowing resin bound tripeptides and dipeptides.

-Leu-Leu-Leu -Leu-Leu-Phe -Leu-Leu-Gly -Leu-Phe-Leu -Leu-Phe-Phe-Leu-Phe-Gly -Leu-Gly-Leu -Leu-Gly-Phe -Leu-Gly-Gly -Phe-Leu-Leu-Leu-Leu-Phe -Phe-Leu-Gly -Phe-Phe-Leu -Phe-Phe-Phe -Phe-Phe-Gly-Phe-Gly-Leu -Phe-Gly-Phe -Phe-Gly-Gly -Gly-Leu-Leu -Gly-Leu-Phe-Gly-Leu-Gly -Gly-Phe-Leu -Gly-Phe-Phe -Gly-Phe-Gly -Gly-Gly-Leu-Gly-Gly-Phe -Gly-Gly-Gly -Leu-Leu -Leu-Phe -Leu-Gly -Phe-Leu -Phe-Phe-Phe-Gly -Gly-Leu -Gly-Phe -Gly-Gly

A rink acid resin having the following structure, wherein “P” representsa polystyrene divinylbenzene resin, was used in the solid phasereactions.

The first N-(9-fluorenylmethoxycarbonyl)-protected amino acid wascoupled to the resin via a bond formation between its C-terminus and thehydroxyl group of the resin using DIPC (Diisopropylcarbodiimide)/DMAP(4-Dimethylaminopyridine). The percent yield was calculated after thecoupling, and if it was below 90%, the coupling was repeated until thepercent yield exceeded 90%. The N-(9-fluorenylmethoxycarbonyl)-group(Fmoc-group) of the first amino acid was removed by treatment with 20%piperidine in 1-methyl-2-Pyrrolidinone (NMP). A Kaiser's Test wasperformed to check the result of the deprotection. The three Kaiser'sreagents (Ninhydrin, Phenol and Potassium Cyanide) were added to analiquot of resin sample to yield a light yellowish solution. The mixturewas heated at 100° C. for 3 minutes, and if the solution turned darkpurple (positive result), the Fmoc-group was considered to have beenremoved. The next amino acid was coupled by peptide bond formation usingDIPC/HOBT(1-Hydroxy-Benzotriazole). NMP was used as the solvent fordeprotection, coupling, and washing and, dichloromethane was used as thedrying reagent. Another Kaiser's test was then performed, with no colorchange (negative result) indicating that the coupling was successful.The coupling reaction was repeated to add additional amino acids.Compound 13 was coupled to the final resin bound amino acid or peptideusing dichloromethane as the solvent. NMP was not used as the solventfor the coupling reaction with compound 13, because NMP has been shownto cause inversion of the chiral center in cysteine, yielding a racemicmixture. Additionally, HOBT was not used in the coupling reaction withcompound 13, because it may also contribute to the formation of amixture of products. The cleavage of the resin from the peptide wasachieved by treatment with 10% acetic acid in dichloromethane. Theproducts were tested by UV and Mass Spectroscopy. HMAF gives two maximalUV absorbances at about 210 nm and 330 nm, so the presence of productswas confirmed by the presence of these two absorbances. MassSpectroscopy was also used to confirm the presence of the desiredproducts.

Using this general procedure, compound 13 was coupled with the 36 tri-and di-peptides shown above, and with the individual amino acids Leu,Phe and Gly, to give a total of 39 compounds of the invention. TheN-acetyl group of these 39 compounds can also be removed usingconditions similar to those described in Example 11 to give anadditional 39 compounds of the invention. Accordingly, in a compound offormula I, R_(x) may preferably represent Leu, Phe, Gly, or any of the36 tri- and di-peptides shown above.

Example 16

Compound 17 (formula I wherein R₁ is(S)-2-(2-carboxypyrrolidin-1-ylcarbonyl)propylthiomethyl; R₂ is methyl;R₃ is hydrogen; R₄ is methyl; R₅ is hydroxy; and R₆ is methyl)

Using a procedure similar to that described in Example 10 exceptreplacing the cysteine used therein with1-(3-mercapto-2-methyl-1-1-oxopropyl)-L-proline (captopril), compound 17was prepared.

Example 17

Compound 18 (formula II wherein R₂ is methyl; R₃ is hydrogen; R₄ ismethyl; R₅ is hydroxy; R₆ is methyl, and R₇ is hydroxymethyl)

Illudin S (178.5 mg) was dissolved in acetic anhydride (2.0 mL) andsodium acetate (96 mg) was added. After 24 hours a mixture of mono anddiacetate was obtained. The monoacetate (171 mg) was disolved indichloromethane and pyridinium dichromate (695 mg) was added. Themixture was stirred at room temperature for 21 hours. Work-up yieldeddehydroilludin S monoacetate (118 mg), which was dissoved in acetone (5mL) and 1M sulfuric acid (3 mL) for 12 hours to give compound 18 (66mg); ¹H NMR (CDCl₃) δ0.63 (1H), 1.13 (1H), 1.23 (4H), 1.38 (4H), 2.05(3H), 3.62 (1H), 3.76 (1H), 682 (1H).

Example 18

The following illustrate representative pharmaceutical dosage forms,containing a compound of formula I or II (‘Compound X’), for therapeuticor prophylactic use in humans.

(I) Tablet 1 mg/tablet ‘Compound X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet ‘Compound X’ 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule ‘Compound X’ 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/ml) mg/ml ‘Compound X’ (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0 N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/ml) mg/ml ‘Compound X’ (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 01 N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can ‘Compound X’ 20.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0

The above formulations may be obtained by conventional procedures wellknown in the pharmaceutical art.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the scope of the invention.

What is claimed is:
 1. A compound of formula I:

wherein R₁ is —(CH₂)_(n)—(X)—(Y); n is 1; X is thio; Y is a 2 to 6membered branched or unbranched carbon chain; wherein said chain issubstituted on carbon with 1, 2, or 3, oxo, heteroaryl; and wherein saidchain may optionally be saturated or unsaturated; R₂ is hydrogen or(C₁-C₆)alkyl; R₃ is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkylthio, aryl, heteroaryl, aryloxy, or heteroaryloxy; R₄ ishydrogen or (C₁-C₆)alkyl; and R₅ is hydroxy, (C₁-C₆)alkoxy, or(C₁-C₆)alkanoyloxy; or R₄ and R₅ taken together are ethylenedioxy; R₆ ishydrogen, carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,halo(C₁-C₆)alkyl, —C(═O)NR_(f)R_(g), or (C₁-C₆)alkyl optionallysubstituted by 1 or 2 hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy,carboxy, or —C(═O)NR_(f)R_(g); R_(a) is hydrogen, (C₁-C₆)alkyl,(C₁-C₆)alkanoyl, phenyl or benzyl; and R_(b), R_(c), R_(f) and R_(g) areeach independently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl orbenzyl; or R_(b) and R_(c) or R_(f) and R_(g), together with thenitrogen to which they are attached, are pyrrolidino, piperidino, ormorpholino; wherein any aryl, heteroaryl, aryloxy, or heteroaryloxy ofY, or R₃ may optionally be substituted by 1, 2, or 3 ((C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, hydroxy (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy,halo, carboxy, mercapto, nitro, or —N(R_(h))(R_(j)); wherein each R_(h)and R_(j) is independently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,phenyl or benzyl; or R_(h) and R_(j) together with the nitrogen to whichthey are attached are pyrrolidino, piperidino, or morpholino; or apharmaceutically acceptable salt thereof.
 2. The compound:

or a pharmaceutically acceptable salt thereof.
 3. A method comprisinginhibiting cancer cells, by contacting said cells with an effectiveamount of a compound of claim
 1. 4. A therapeutic method comprisingtreating cancer by administering an effective amount of a compound ofclaim 1 to a mammal in need of such therapy.
 5. A pharmaceuticalcomposition comprising one or more compounds of claim 1 in combinationwith a pharmaceutically acceptable diluent or carrier.